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Functional Study on the Genes in Biosynthetic Pathway of an Aromatic Polyketide Antibiotic Medermycin

Author: CaiXiaoFeng
Tutor: LiAiYing
School: Central China Normal University
Course: Microbiology
Keywords: Medermycin Biosynthetic pathway Streptomyces gene knock-out and complementation Gene co-expression Natural product analysis
CLC: Q93
Type: Master's thesis
Year: 2009
Downloads: 44
Quote: 1
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Abstract


Medermycin is a streptomyces aromatic polyketide antibiotic,and shows strong antibacterial and antitumor activity.The biosynthetic pathway of medermycin has yet to be clarified.In the present study,functional analysis of several genes located in the medermycin biosynthetic gene cluster was carried out by bioinformatics,genetics,analytical chemistry and other approaches:（1） A novel C-glycosyltransferase gene:med-ORF8,a putative glycosyltransferase gene,is proposed to be involved in the rare C-glycosylation in the biosynthesis of medermycin according to homology analysis.In order to investigate the function of med-ORF8,firstly,gene inactivation in vivo was carried out by RedirectTM technology to gain a mutant gene cluster, which contains all necessary genes for biosynthesis of medermycin except med-ORF8. Heterologous expression of this mutant gene cluster resulted in the absence of medermycin production.Meanwhile,the normal production of medermycin could be restored by complementation with med-ORF8.Data from LC-MS measurement suggested that med-ORF8 is an essential gene in the pathway of medermycin biosynthesis.（2） Glycosylsynthase genes:six glycosylsynthase-homologous genes in medermycin gene cluster are proposed to be involved in the rare angolosamine（deoxyhexose,DOH） biosynthesis. pAYT55,a co-expression plasmid containing these 6 putative DOH sugar biosynthetic genes and med-ORF8 was transformed into Streptomyces coelicolor B135,a producer of kalafungin which is a putative final intermediate without angolosamine attachment.LC-MS measurement proved that kalafungin was glycosylated in the resulting recombinant strain and converted to medermycin.The present data suggested that six putative glycosylsynthase genes together with med-ORF8 are involved in and eonough for glycosylsynthesis and glycosyltransferring in the medermycin biosynthesis.（3） A novel transcriptional regulatory gene:med-ORF10 is proposed to be a positive transcriptional regulatory gene in the biosynthesis of medermycin.In order to prove the function of med-ORF10,pAYT64 containing a mutant gene cluster gained with the same strategy as above for gene inactivation of med-ORF10 was transformed into a heterologous host Streptomyces coelicolor CH999.LC-MS analysis suggested the med-ORF10 disruptant produced reduced amount of medermycin,together with accumulation of a shunt product. These results provided some evidences for the function of med-ORF10 as a positive regulatory gene.Our data here opened up the possibility to clarify extensively the biosynthetic pathway of medermycin.

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